This R01 application entitled Lead Optimization of Novel Inhibitors of Tissue Non-specific Alkaline Phosphatase is in response to PAR-12-060 Solicitation of Validated Hits for the Discovery of in vivo Chemical Probes. Medial vascular calcification (MVC) is the major cause of morbidity and mortality in generalized arterial calcification of infancy (GACI), a severe childhood disease, and contributes to cardiovascular deterioration in Kawasaki disease (KD), chronic kidney disease (CKD), as well as in diabetes, obesity and aging. The relevance of the mineralization inhibitor extracellular inorganic pyrophosphate (ePPi) and its homeostasis in these conditions has been clearly established. Reduced levels of ePPi have been linked to elevated expression levels of tissue non-specific alkaline phosphatase (TNAP), which hydrolizes ePPi, thus eliminating its inhibitory effect in tissue stricken by MVC. For example, we have recently observed an upregulation of TNAP in vascular smooth muscle cells (VSMC) and also in uremic aortas, suggesting that excessive TNAP activity is an important cause of ePPi deficiency and medial calcification. We hypothesized that potent small molecule TNAP inhibitors are likely to cause a reduction in TNAP activity following systemic administration, resulting in an increase in the local amount of ePPi to prevent or ameliorate vascular calcification. Therefore, we propose a strategy for increasing ePPi levels by reducing TNAP activity with small molecule TNAP inhibitors. We recently reported the characterization and preliminary optimization of arylsulfonamide-derived inhibitors of TNAP. These compounds, which function via an uncompetitive mechanism, are ready for full-scale chemistry optimization to provide lead compounds ready for in vivo proof- of-concept studies. Therefore our Specific Aims are: 1) Design and synthesize optimized TNAP inhibitors that are orally active in vivo; 2) Assess the potency and selectivity of TNAP inhibitors in relevan in vitro assays; 3) Evaluate novel small molecule TNAP inhibitors using in vitro ADME/T and in vivo pharmacokinetic (PK) assays; and 4) Characterize lead TNAP inhibitor probes in rodent ex vivo and in vivo models of vascular calcification. The TNAP inhibitors generated will provide powerful tools for testing the hypothesis that enhancing ePPi levels by modulating TNAP activity protects against MVC, while laying a foundation for future development of a novel class of medications for the treatment of the family of diseases caused by MVC.